A new synthesis of isoaurones: Cytotoxic activity of compounds related to the alleged structure of isoaurostatin

Article abstract of DOI:10.1016/j.bmcl.2005.06.045

A new synthesis of isoaurones related to the alleged structure of isoaurostatin, via Heck intramolecular cyclization of cinnamic esters of 2-iodophenols, is reported. The cytotoxic activity of these isoaurones is lower than that of the structurally very similar 4-arylcoumarins.

Full text of DOI:10.1016/j.bmcl.2005.06.045

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4313–4316
A new synthesis of isoaurones: Cytotoxic activity of compounds
related to the alleged structure of isoaurostatin
Eleonora Rizzi,a Sabrina Dallavalle,a Lucio Merlini,a,* Giovanni Luca Beretta,b
Graziella Pratesib and Franco Zuninob
a
`
Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2, I 20133 Milano, Italy
Unita di Farmacologia Preclinica, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian, I 20133 Milano, Italy
b
`
Received 22 March 2005; revised 15 June 2005; accepted 16 June 2005
Abstract—A new synthesis of isoaurones related to the alleged structure of isoaurostatin, via Heck intramolecular cyclization of
cinnamic esters of 2-iodophenols, is reported. The cytotoxic activity of these isoaurones is lower than that of the structurally very
similar 4-arylcoumarins.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
activity was the similarity of their structure with that of
combretastatins,4 of which isoaurones can be considered
rigidified analogues. Most recently, however, when this
work was already in a final stage, the revised structure
2, corresponding to the isoflavone daidzein, was put for-
ward for isoaurostatin by Venkateswarlu et al.,5 on the
basis of the divergence of the spectroscopic data report-
ed by Suzuki et al.1 for isoaurostatin from those for the
synthetic compound 1, and the similarity with those for
daidzein 2.
Isoaurostatin is a metabolite of the fungus Thermomo-
nospora alba, strain no. 1520, that was isolated by Suzuki
et al. in 2001 and to which the structure of E-6-hydroxy-
3-(4-hydroxybenzylidene)-benzo[b]furan-2-one (1) was
attributed on the basis of spectroscopic analysis.1
This publication prompts us to disclose our own results,
that is, a new method of synthesis of isoaurones and
data on the cytotoxic activity of a series of compounds
related to structure 1.
The report by the same authors that isoaurostatin inhib-
ited the relaxation activity of calf thymus topoisomerase
I in a competitive manner, and therefore, with a mecha-
nism different from that of camptothecin and of its ana-
logues, attracted our attention. As we are interested in
the synthesis of new inhibitors of topoisomerase I as po-
tential antitumor agents,2,3 we undertook the synthesis
of some analogues of isoaurostatin to investigate their
cytotoxic potential. Another feature of these compounds
that could justify the interest for their possible cytotoxic
2. Results and discussion
Isoaurones are extremely rare as natural compounds,
and only two of them have been reported so far.6,7
Therefore, there are also few methods of synthesis of
this class of compounds. An example of cyclization of
the appropriate hydroxyacid prepared by Aryl-Zn aryla-
tion of 2,3-dibromopropenoate was reported by Rossi
et al.8 Condensation of aromatic aldehydes with lac-
tones of 2-hydroxyphenylacetic acids9 or with the acids
themselves10 in the presence of acetic anhydride has
been reported, and the former method has been used
for the recent synthesis of 1 itself.5 In another method,
Keywords: Isoaurones; Arylcoumarins; Antitumor activity.
*
Corresponding author. Tel.: +39 0250316812; fax: +39
0250316801; e-mail: lucio.merlini@unimi.it
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.045

4314
E. Rizzi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4313–4316
phenols are condensed with phenylpyruvic acids.11 Most
of these methods are not versatile, inasmuch they re-
quire the synthesis of the appropriately substituted
phenylacetic acids, and, moreover, in our hands they
gave erratic results, so that we devised a new synthesis
of the isoaurone skeleton, based on an intramolecular
Heck reaction.
as IC50 inhibitory concentrations, show that the cytotox-
icity of the prepared isoaurones is modest.
The cytotoxic activity of this series of compounds is
comparable to that of corresponding isomeric 4-aryl-
coumarins against the CEM human leukemia cell line,
recently reported by Bailly et al.16 However, in the
4-arylcoumarin series, compound 7, with the 3-hy-
droxy-4-methoxy substitution in the 4-aryl group,
showed a potent activity (0.083 lM). This substitution
is particularly favorable in the combretastatin series,
compare to combretastatin A-4.21
Esterification of ortho-iodophenols (3) with cinnamic
acids (4) gave the corresponding esters (5) that were sub-
jected to Heck reaction in acetonitrile in the presence of
Pd(OAc)2 with NaOAc and BuN4Br, or Pd(OAc)2 with
tri-(o-tolyl)phosphine and triethylamine. The reaction
gave the expected 3-benzylidenebenzo[b]furan-2-ones in
good yield as mixtures of E and Z isomers (Scheme 1).
This result is at variance with respect to the condensa-
tion of aryl aldehydes with benzo[b]furan-2-ones that
gives predominantly Z isomers.12
The correctness of the structures of the synthesized com-
pounds appears from the spectroscopic data, in particu-
lar H NMR, that are consistent with those of known
Therefore, we synthesized compound 6f, with the same
substitutions as well as 7. Moreover, to make a sound
comparison of the biological data, we synthesized com-
pound 7 that was tested on the same H460 cell line as
for compound 6f. However, in spite of the strong sim-
ilarity of the two compounds (Fig. 1 shows the overlay
of the MM2 energy-minimized structures of E-6f and 7,
which indicates a remarkable superimposition of the
oxygen functions of the two compounds; the distances
1
isoaurones.14 Moreover, the compounds are different
from the isomeric 4-arylcoumarins that show a typical
chemical shift of the vinylic proton around 6.0–
6.3 ppm,15,16 whereas the same proton in our com-
pounds is around 7.5–7.8 ppm. As for the assignment
of Z versus E configuration, there is some uncertainty
in the literature, most authors basing their assignment
on the analogy with reports of preceding papers. Perusal
of the literature showed that in two studies the configu-
ration was assigned on the basis of further evidence
other than just the value of the chemical shift of the
vinylic proton. These studies by Da¨twyler et al.,17 who
used the value of the coupling constant between the car-
bonyl carbon and the vinylic proton, and by Gadre and
Marathe,18 who used the lanthanide-induced shift meth-
od, indicated that the vinylic proton of the Z form lies
around 7.5 ppm, whereas that of the E form lies around
7.7–7.9 ppm. In fact most of the other literature is con-
sistent with this criterion. Ironically enough, on this ba-
sis, the assignment by Venkateswarlu et al.5 of the E and
Z structures to the isomers of compound 1 with chemi-
cal shift of 7.50 and 7.72 ppm, respectively, must be
reversed.19
˚
between the oxygens of each pair being <0.7 A), the
isoaurone 6f appeared about 70-fold less active than
the arylcoumarin 7. Therefore, subtle structural differ-
ences must play a role in the cytotoxic activity of these
compounds.
3. Experimental
3.1. 2-Iodo-3,5-dimethoxyphenol
To a solution of 3,5-dimethoxyphenol (1 equiv) in
Et2O (about 0.2 M), a saturated solution of NaHCO3
was added and this mixture was vigorously stirred in
N2 atmosphere. A solution of ICl (1.5 equiv) in Et2O
was added dropwise and the mixture was stirred at rt
for 2 h. After completion of the reaction, monitored
by TLC (hexane/EtOAc, 8:2), the organic layer was
washed with a 1 M solution of Na2S2O3 (3 · 10 mL)
and dried over Na2SO4. The crude product was puri-
fied by flash column chromatography on silica gel (hex-
ane/EtOAc, 9:1) to give 0.945 g (52%) of the product
(mp 71–72 ꢁC).
The compounds 6a–g were evaluated for their cytotoxic-
ity against the human non-small lung carcinoma cell line
H460, using topotecan as a reference compound. This
cell model was chosen for its sensitivity to topoisomer-
ase I inhibitors, likely related to the overexpression of
the target enzyme.20 The results, indicated in Table 1
Scheme 1. Synthesis of isoaurones.

E. Rizzi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4313–4316
Table 1. Yields and cytotoxic activity of isoaurones
4315
Compound
R1
R2
R3
R4
R5
R6
Isomera
Method
Yield of 5 (%)
Yield of 6 (%)
IC50(lM)
>39
37.7
>30
10.1
6a
6b
6c
6db
6e
6f
H
H
H
OMe
OMe
OBz
OH
H
H
E
A
B
B
75
86
60
84
75
60
H
H
OMe
H
OMe
H
H
E
H
H
H
E/Z 4:1
E/Z 4:1
E/Z 1:1
E/Z 1:1
E/Z 3:1
H
H
H
H
H
H
H
H
OMe
OMe
OAc
OH
OH
H
H
B
B
54
21
64
37
9.3
8.8
OMe
H
OMe
H
H
H
OAc
6gc
7
H
>50
0.12
a Ratio estimated on the basis of the NMR spectra.
b Obtained from 6c by debenzylation (70% yield) with chlorosulfonylisocyanate.13
c Prepared by condensation of 2,5-dihydroxyphenylacetic acid lactone with 4-hydroxybenzaldehyde in the presence of acetic anhydride.
(1 equiv), AcONa (1.5 equiv) (method A) or P(o-Tol)3
(40% mol) and Et3N (1.4 equiv) (method B) were added.
The mixture was stirred and refluxed in Ar atmosphere
to a complete disappearance of the ester (TLC hexane/
EtOAc 7:3). After evaporation of solvent, the crude
product was taken up with EtOAc, washed with water
(3 · 10 mL), dried over Na2SO4, and purified by flash
column chromatography on silica gel (hexane/EtOAc,
8:2 or 9:1).
3.4. Compound 6g
To a solution of 2,5-dihydroxyphenylacetic acid lactone
(0.1 g, 0.66 mmol) in acetic anhydride (2.5 mL),
4-hydroxybenzaldehyde (0.081 g, 0.66 mmol) was
added. This mixture was stirred and refluxed in Ar
atmosphere for 3 h. After completion of the reaction
(TLC hexane/EtOAc, 6:4), the solvent was removed in
vacuo, and the crude product was taken up with EtOAc
(10 mL), washed with water (3 · 5 mL), dried over
Na2SO4, and purified by flash chromatography on silica
gel (hexane/EtOAc, 7:3) to give 0.051 g, yield 24%.
Figure 1. Overlay of the MM2 energy-minimized structures of E-6f
(white) and 7 (black).
3.5. Assessment of antitumor activity
3.2. General procedure for the preparation of cinnamic
esters
Cells were cultured in RPMI-1640 containing 10% fetal
calf serum. Cytotoxicity was assessed by growth inhibi-
tion assay after 1 h drug exposure. Cells in the logarith-
mic phase of growth were harvested and seeded in
duplicates into six-well plates. Twenty-four hours after
seeding, cells were exposed to the drug and harvested
72 h after exposure and counted with a Coulter counter.
IC50 is defined as the inhibitory drug concentration
causing a 50% decrease of cell growth over that of
untreated control. All compounds were dissolved in
DMSO prior to dilution into the biological assay.
To a suspension of the appropriate cinnamic acid
(1 equiv) in dry CH2Cl2 (about 0.1 M), DCC (1.2
equiv) and DMAP (catalytic) were added. The mixture
was stirred and refluxed for 1 h in N2 atmosphere.
The formation of the activated acid was monitored
by TLC (EtOAc/hexane, 6:4). The phenol (1.1 equiv)
was added and the reaction was stirred and refluxed
to complete disappearance of the intermediate (TLC
hexane/EtOAc, 6:4). After completion of the reaction,
the mixture was filtered and the solvent was removed
in vacuo. The crude product was purified by flash
column chromatography on silica gel with hexane/
EtOAc, 8:2.
Acknowledgments
5a: mp 92–94 ꢁC; 5b: mp 142–144 ꢁC; 5e: mp 110 ꢁC; 5c:
mp 112–115 ꢁC; 5f: mp 127–128 ꢁC.
We are indebted to MIUR (Programs Prin 2003 and
FIRB 2001) for financial support.
3.3. General procedure for the preparation of 3-ben-
zylidenebenzo[b]furan-2-ones
References and notes
To a solution of the cinnamic ester (1 equiv) in CH3CN
(about 0.1 M) Pd(OAc)2 (5–10% mol) and n-Bu4NBr
1. Suzuki, K.; Yahara, S.; Maehata, K.; Uyeda, M. J. Nat.
Prod. 2001, 64, 204.

4316
E. Rizzi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4313–4316
dd, H7, J = 7.73 Hz, 1.42 Hz), 7.05 (1H, ddd, H6, J = 7.73,
2. Dallavalle, S.; Merlini, L.; Morini, G.; Musso, L.; Penco,
0
0
S.; Beretta, G. L.; Tinelli, S.; Zunino, F. Eur. J. Med.
Chem. 2004, 39, 507.
3. Dallavalle, S.; Merlini, L.; Beretta, G. L.; Tinelli, S.;
Zunino, F. Bioorg. Med. Chem. Lett. 2004, 14,
5757.
4. (a) Cirla, A.; Mann, J. Nat. Prod. Rep. 2003, 558; (b) Nam,
N. H. Curr. Med. Chem. 2003, 10, 1697.
5. (a) Venkateswarlu, S.; Panchagnula, G. K.; Guraiah, M.
B.; Subbaraju, G. V. Tetrahedron 2005, 61, 3013; (b) The
same revision has been proposed by Ghisalberti, E. L. Nat.
Prod. Res. 2005, 19, 453.
7.73, and 1.42 Hz), 6.95 (2H, d, H3 and H5 , J = 8.14 Hz).
6e: isomer Z: 1H NMR (CDCl3) d 9.86 (1H, d, H4,
J = 7.82 Hz), 7.77 (1H, s, CH@), 7.32 (1H, dd, H6,
0
J = 7.82 and 7.82 Hz), 7.32 (1H, d, H2 , J = 1.86 Hz), 7.25
0
(1H, dd, H6 , J = 8.56 and 1.86 Hz), 7.13 (1H, d, H7,
J = 7.82 Hz), 7.06 (1H, dd, H5, J = 7.82 and 7.82 Hz), 6.95
0
(1H, d, H5 , J = 8.56 Hz), 5.75 (1H, br s, OH), 3.99 (3H, s,
OCH3). Isomer E: H NMR (CDCl3) d 7.89 (1H, dd, H4,
J = 7.82 and 1.49 Hz), 7.51 (1H, s, CH@), 7.50 (1H, dd, H6 ,
J = 7.82 and 1.84 Hz), 7.30 (1H, ddd, H6, J = 7.82, 7.82, and
0
1.49 Hz), 7.24 (1H, dd, H2 , J = 1.84 Hz), 7.15 (1H, ddd, H5,
1
0
6. Pelter, A.; Ward, R. S.; Gray, T. I. J. Chem. Soc., Perkin
Trans. 1 1976, 2475.
7. Schildknecht, H.; Kornig, W.; Siewerdt, R.; Krauss, D.
Liebigs Ann. Chem. 1970, 734, 116.
8. Rossi, R.; Bellina, F.; Carpita, F.; Gori, R. Gazz. Chim.
Ital. 1995, 125, 381.
9. Barbier, M. Liebigs Ann. Chem. 1987, 545.
10. Czaplicki, S.; von Kostanecki, S.; Lampe, V. Ber. Dtsch.
Chem. Ges. 1909, 42, 834.
J = 7.82, 7.82, and 1.49 Hz), 7.08 (1H, d, H7, J = 7.82 Hz),
0
6.94 (1H, d, H5 , J = 8.93 Hz), 3.99 (OCH3). MS: m/z 268
(M+, 100), 197 (20). 6f: isomer Z: 1H NMR (CDCl3) d 7.67
0
(1H, s, CH@), 6.97 (1H, dd, H6 , J = 8.19 and 2.23 Hz), 6.93
0
0
(1H, d, H2 , J = 2.23 Hz), 6.84 (1H, d, H5 , J = 8.19 Hz),
6.39 (1H, d, H7, J = 2.23 Hz), 6.20 (1H, d, H5, J = 2.23 Hz),
5.50 (1H, br s, OH), 3.96 (3H, s, OCH3), 3.84 (3H, s, OCH3),
3.65 (3H, s, OCH3). Isomer E: 1H NMR (CDCl3) d 7.90 (1H,
0
s, CH@), 7.73 (1H, dd, H6 , J = 8.19 and 2.23 Hz), 7.70 (1H,
0
0
11. Van der Westhuizen, J. H.; Ferreira, D.; Roux, D. G.
J. Chem. Soc., Perkin Trans. 1 1977, 1517.
12. Barbier, M. Heterocycles 1988, 27, 955.
13. Kim, J. D.; Han, G.; Zee, O. P.; Jung, Y. H. Tetrahedron
Lett. 2003, 44, 733.
d, H2 , J = 2.23 Hz), 6.88 (1H, d, H5 , J = 8.19 Hz), 6.31
(1H, d, H7, J = 2.23 Hz), 6.25 (1H, d, H5, J = 2.23 Hz), 5.50
(1H, br s, OH), 3.95 (3H, s, OCH3), 3.94 (3H, s, OCH3), 3.83
(3H, s, OCH3). MS: m/z 327 (Mꢀ1)+, 100. 6g: isomer Z: 1H
0
0
NMR (CDCl3) d 8.24 (2H, d, H2 and H6 , J = 8.56 Hz), 7.54
14. Spectroscopic data: E-6a: mp 130 ꢁC. 1H NMR (CDCl3) d
7.81 (1H, dd, H4, J = 7.82 and 1.12 Hz), 7.81 (1H, s, CH@),
(1H, s, CH@), 7.30 (1H, d, H4, J = 2.23 Hz), 7.24 (2H, d, H3
0
and H5 ,J = 8.56 Hz), 7.14 (1H, d, H7, J = 8.56 Hz), 7.05
0
0
0
7.69 (2H, d, H2 and H6 , J = 8.93 Hz), 7.32 (1H, ddd, H5,
J = 7.82, 7.82, and 1.12 Hz), 7.13 (1H, dd, H7, J = 7.82 and
1.12 Hz), 7.05 (1H, ddd, H6, J = 7.82, 7.82, and 1.12 Hz),
(1H, dd, H6, J = 8.56 Hz, J = 2.23 Hz), 2.33 (3H, s, OAc),
2.32 (3H, s, OAc). Isomer E: 1H NMR (CDCl3) d 7.85 (1H,
0
0
s, CH@), 7.69 (2H, d, H2 and H6 , J = 8.56 Hz), 7.45 (1H, d,
0
0
0
0
7.00 (2H, d, H3 and H5 , J = 8.93 Hz), 3.80 (3H, s, OCH3);
MS: m/z 252 (M+, 100), 209 (20). E-6b: mp 183–185 ꢁC. 1H
NMR (CDCl3) d 7.86 (1H, dd, H4, J = 7.82 and 1.49 Hz),
7.79 (1H, s, CH@), 7.34 (1H, ddd, H6, J = 7.82, 7.82, and
1.49 Hz), 7.15 (1H, dd, H7, J = 7.82 and 1.49 Hz), 7.06 (1H,
H4, J = 2.23 Hz), 7.25 (2H, d, H3 and H5 , J = 8.56 Hz),
7.14 (1H, d, H7, J = 8.56 Hz), 7.05 (1H, dd, H6, J = 8.56 Hz,
J = 2.23 Hz), 2.34 (3H, s, OAc), 2.27 (3H, s, OAc).
15. Jia, C.; Piao, D.; Kitamura, T.; Fujiwara, Y. J. Org.
Chem. 2000, 65, 7516.
0
ddd, H5, J = 7.82, 1.49, and 1.49 Hz), 6.93 (2H, s, H2 and
16. Bailly, C.; Bal, C.; Barbier, P.; Combes, S.; Finet, J. P.;
Hildebrand, M. P.; Peyrot, V.; Wattez, N. J. Med. Chem.
2003, 45, 5437.
17. Da¨twyler, P.; Bosshardt, H.; Bernhard, H. O.; Hesse, M.;
Johne, S. Helv. Chim. Acta 1978, 61, 2646.
18. Gadre, S. Y.; Marathe, K. G. Synth. Commun. 1988, 18,
1015.
19. As Venkateswarlu et al. used as a reference the data by
Marathe et al.22 who named ÔtransÕ the Z form, and ‘‘cis’’
the E form, it is possible that they were misled by this
nomenclature.
0
H6 ), 3.94 (3H, s, OCH3), 3.88 (6H, s, OCH3). MS: m/z 312
(M+, 100), 297 (48). 6c: isomer Z: 1H NMR (CDCl3) d 8.26
0
0
(2H, d, H2 and H6 , J = 8.93 Hz), 7.55 (1H, s, CH@), 7.50
(1H, ddd, H5, J = 7.82, 7.82, and 1.42 Hz), 7.48–7.01 (10H,
m, 10Ar), 0.15 (2H, s, OCH2Ar). Isomer E: 1H NMR
0
(CDCl3) d 7.82 (2H, m, CH@ + H4), 7.68 (2H, d, H2 and
0
H6 , J = 8.93 Hz), 7.33 (1H, ddd, H5, J = 8.19, 8.19, and
1.49 Hz), 7.29–7.48 (6H, m, 6Ar), 7.12 (1H, dd, H7, J = 8.19
0
0
and 1.49 Hz), 7.09 (2H, d, H3 and H5 , J = 8.93 Hz), 5.15
(2H, s, OCH2Ar). MS: m/z 328 (M+, 20), 91 (100). 6d: isomer
Z: 1H NMR (CDCl3) d 8.23 (2H, d, H2 and H6 ,
J = 8.54 Hz), 7.78 (1H, dd, H4, J = 7.73 and 1.42 Hz), 7.55
(1H, s, CH@), 7.51 (1H, ddd, H5, J = 7.73, 7.73, and
1.42 Hz), 7.30 (1H, ddd, H6, J = 7.73, 7.73, and 1.42 Hz),
20. Giaccone, G.; Gazdar, A. F.; Beck, H.; Zunino, F.;
Capranico, G. Cancer Res. 1992, 52, 1666.
0
0
´
21. Maya, A. B. S.; Perez-Melero, C.; Mateo, C.; Alonso, D.;
´
´
Fernandez, J. L.; Gajate, C.; Mollinedo, F.; Pelaez, R.;
Caballero, E.; Medarde, M. J. Med. Chem. 2005, 48, 556,
and references quoted therein.
0
7.16 (1H, dd, H7, J = 7.73 and 1.42 Hz), 6.94 (2H, d, H3 and
1
0
H5 , J = 8.14 Hz). Isomer E: H NMR (CDCl3) d 7.79–7.80
0
0
(2H, m, H4 + CH@), 7.64 (2H, d, H2 and H6 , J = 8.14 Hz),
7.32 (1H, ddd, H5, J = 7.73, 7.73, and 1.42 Hz), 7.15 (1H,
22. Marathe, K. G.; Byrne, M. J.; Vidwans, R. Tetrahedron
1966, 22, 1789.